Development of a novel 96-microwell assay with high throughput for determination of olmesartan medoxomil in its tablets
© Darwish et al 2010
Received: 9 October 2011
Accepted: 3 January 2012
Published: 3 January 2012
A novel 96-microwell-based spectrophotometric assay has been developed and validated for determination of olmesartan medoxomil (OLM) in tablets. The formation of a colored charge-transfer (CT) complex between OLM as a n-electron donor and 2, 5-dichloro-3, 6-dihydroxy-1, 4-benzoquinone (p-chloranilic acid, pCA) as a π-electron acceptor was investigated, for the first time, and employed as a basis in the development of the proposed assay. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 490 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 1-200 μg ml-1. The limits of detection and quantitation were 0.3 and 1 μg ml-1, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from hydrochlorothiazide and amlodipine that are co-formulated with OLM in some formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has great practical value in the routine analysis of OLM in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for OLM, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.
KeywordsOlmesartan medoxomil Charge-transfer reaction Spectrophotometry Microwell-based assay High throughput analysis
OLM has not yet been officially described in any pharmacopoeia. A literature survey revealed that several analytical methods were reported for its determination. These methods include high-performance thin-layer chromatography , liquid chromatography [3–9], and capillary zone electrophoresis . These methods were not simple to perform, time-consuming, and utilize expensive instruments that are not available in most quality control laboratories. In general, spectrophotometry is the most widely used technique in pharmaceutical analysis because of its inherent simplicity and wide availability in most quality control laboratories . However, few spectrophotometric methods have been reported for the analysis of OLM in its pharmaceutical tablets [12–15]. Unfortunately, these methods suffer from major drawbacks such as decreased selectivity due to measuring the native light absorption of OLM in the blue-shifted ultraviolet region, which might be subjected to interferences [12–14]. Besides the tedious liquid-liquid extraction procedures using large volumes of organic solvents in the methods based on formation of ion-pair associates . Therefore, the development of a new alternative spectrophotometric method for determination of OLM in pharmaceutical formulations is very essential.
The molecular interactions between the electron-donating pharmaceutical compounds and electron-accepting reagents are generally associated with the formation of intensely colored CT complexes, which usually absorb radiations in the visible region. The rapid formation of these complexes leads to their widespread utility in the development of visible spectrophotometric methods for analysis of many pharmaceutical compounds [16–20]. Literature survey revealed that the CT reaction of OLM has not been investigated yet. This fact promoted our interest in employment of the CT-reaction as a basis for the development of a new spectrophotometric method for determination of OLM. However, all the conventional CT-based spectrophotometric methods that have been reported so far are not automated and consequently their throughput is low, thus their applications in pharmaceutical quality control laboratories are limited. Moreover, these methods suffer from the consumption of large volumes of organic solvents, which leads to high analysis cost, and more importantly, the incidence of exposure of the analysts to the toxic effects of the organic solvents [21–25].
For these reasons, the present study is devoted to investigate the CT reaction of OLM, and its employment in the development of a novel non-conventional spectrophotometric assay with high analysis throughput and can reduce the consumption of organic solvents in the determination of OLM in tablets.
Microwell-plate absorbance reader (ELx 808, Bio-Tek Instruments Inc. Winooski, USA) was used for all the measurements in 96-microwell plates. UV-1601 PC (Shimadzu, Kyoto, Japan) ultraviolet-visible spectrophotometer with matched 1 cm quartz cells was used for recording the absorption spectra. 96-Microwell plates were a product of Corning/Costar Inc. (Cambridge, USA). Finnpipette adjustable 8-channel-pipette was obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Chemicals and tablets
OLM was obtained from AK Scientific Inc. (California, USA). Amlodipine besylate and hydrochlorothiazide were obtained from Sigma Chemical Co. (St. Louis, USA). pCA (BDH Chemicals, Poole, UK) was 0.5% (w/v) in acetonitrile and it was prepared fresh daily. Olmetec® tablets (SAJA Pharmaceuticals, Jeddah, Saudi Arabia) labeled to contain 20 mg OLM were obtained from the local market.
Preparation of standard and tablet solutions
Preparation of stock standard solutions
Into a 5-ml calibrated flask, 10 mg of OLM was accurately weighed, dissolved in 2 ml methanol, and completed to volume with the same solvent. This stock solution was diluted with methanol to obtain the suitable concentrations that lie in the linear range of the assay.
Preparation of tablet sample solutions
Twenty tablets were weighed and finely powdered. A quantity of the powder equivalent to 20 mg of OLM was transferred into a 10-ml calibrated flask, dissolved in 4 ml methanol, swirled and sonicated for 5 min, completed to volume with the methanol, shaken well for 15 min, and filtered. The first portion of the filtrate was rejected, and a measured volume of the filtrate was diluted quantitatively with methanol to yield the suitable concentrations that lie in the linear range of the assay.
General analytical procedure
Accurately measured aliquots (100 μl) of the standard or sample solution containing varying amounts of OLM (1-200 μg ml-1) were transferred into wells of 96-microwell assay plates. One hundred microliters of pCA solution (0.5%, w/v) was added, and the reaction was allowed to proceed at room temperature (25 ± 1°C) for 5 min. The absorbances of the resulting solutions were measured at 490 nm by the microwell-plate reader. Blank wells were treated similarly except 100 μl of methanol was used instead of sample, and the absorbances of the blank wells were subtracted from those of the other wells.
Determination of molar ratio
The Job's method of continuous variation  was employed. Master equimolar solutions (2 × 10-3 M) of each of OLM and pCA were prepared. Series of 200 μl portions of the master solutions of OLM with pCA were made up comprising different complementary ratios (0:10, 1:9, ........., 9:1, 10:0, inclusive) in each well of the 96-microwell assay plate. The reaction was allowed to proceed at room temperature (25 ± 1°C) for 5 min. The absorbances of the developed colors were measured at 490 nm by the microwell-plate reader against blank wells treated similarly except methanol was used instead of OLM sample. The measured absorbances were plotted as a function of OLM mole fraction. The generated plot was used for determination the molar ration of OLM:pCA.
Molecular modeling for the CT complex of OLM with pCA
The molecular modeling for the CT complex was performed by using CS Chem3D Ultra, version 9 (Cambridge Soft Corporation, Cambridge, MA, USA) implemented with molecular orbital computations software (MOPAC), and molecular dynamics computations software (MM2).
Results and discussion
Strategy for assay development and its design
In the present study, OLM was selected based on its therapeutic importance, clinical success, and the expected electron-donating ability. Previous studies involving CT reactions with polyhalo-/polycyanoquinone electron n-acceptors revealed that pCA is one of the most efficient reagents in terms of its reactivity [18, 27]. Furthermore, its CT reaction with electron-donating analytes is instantaneous [18, 19]. For these reasons, pCA was used as electron acceptor in the development of the proposed assay. The proposed assay was designed to employ 96-microwell assay plate as the CT reaction was carried out in microwells of the assay plate (200-μl reaction volume). The solutions were dispensed by 8-channel pipette, and the absorbances of the colored CT complex were measured by 96-microwell-plate absorbance reader. The 96-microwell design of the proposed assay was based on the previous success of Darwish et al  in the utility of this design for determination of some other pharmaceuticals.
Reaction and spectral characteristics
Since the interaction of OLM with pCA in acetonitrile gave a violet product, it might be concluded that HA- was the form of pCA involved in the reaction described herein.
Optimization of experimental conditions
Optimum conditions for the charge-transfer reaction of OLM with PCA
pCA conc. (%, w/v)
0.1 - 2
Reaction time (min)
0 - 30
25 - 60
400 - 600
Molar ratio of the reaction, molecular modeling, and proposing the site of interaction
Validation of the proposed assay
Linearity and sensitivity
Quantitative parameters for the analysis of OLM by the proposed assay
Range (μg ml-1)
LOD (μg ml-1)
LOQ (μg ml-1)
Accuracy and precision
Accuracy of the proposed assay was assessed by analytical recovery studies. Recovery was determined by the standard addition method. Known amounts of OLM were added to pre-determined drug-containing tablets, and then determined by the proposed assay. The mean analytical recovery was calculated and found to be 97.6-99.7 ± 0.5-1.5% indicating the accuracy of the proposed assay.
Precision of the proposed assay at different OLM concentrations
Concentration (μg ml-1)
Relative standard deviation
Within-assay, n = 5
Between-assays, n = 5
This high level of precision was attributed to the accuracy of the volumes that have been concomitantly dispensed in the microwells by multi-channel pipettes, and completeness of the reaction the small volume (200 μl).
The proposed assay has the advantages that the measurements are performed in the visible region, away from the UV-absorbing interfering substances that might be co-extracted from dosage forms that contain OLM. The interference from the congenital drugs that is co-formulated with OLM in some dosage forms was studied. These drugs were amlodipine besylate [33–35] hydrochlorothaizde [35, 36] and the chemical structures of these drugs are given in Figure 1. Potential interferences of these drugs were studied in a ratio which is normally present in their combined dosage forms. No interferences from these drugs were found with OLM in the proposed assay.
This specificity of the CT reaction for OLM was attributed to its basic character, which allows the CT, rather than hydrochlorothaizde, that does not have sufficient basicity to achieve CT reaction (pKa = 7.9) . Although, amlodipine has basic character, however it is co-formulated with OLM as besylate salt that did not show any ability for CT reaction with pCA under the proposed assay conditions. As well, no interference was observed from the excipients with the proposed assay as indicated from the obtained good recovery (mentioned above). The absence of interference from the excipients, even though they contain basic component(s) was attributed to the extraction of the OLM tablets prior to the analysis with methanol in which the excipients do not dissolve.
Application of the proposed assay in the analysis of OLM tablets
Analysis of OLM in tablets by the reported and proposed methods
Content (% ± SD)a
99.8 ± 1.24
100.2 ± 0.9
The present study described the development and validation of a novel microwell-based spectrophotometric assay for the determination of OLM based on its CT reaction with pCA reagent. In this assay, the CT reaction was carried out in 96-microwell plates (200-μl reaction volume) instead of the conventional volumetric flasks (10,000-μl volume). The absorbances were measured by microwell-plate reader instead of the conventional spectrophotometer. The assay described herein offered the following advantages:
– Reduction in the consumption of organic solvents in the CT-based spectrophotometric analysis, accordingly reduction in the exposures of the analysts to the toxic effects of organic solvents.
– Reduction in the analysis cost by 50-folds which can be reflected on the price for the finished dosage forms, thus it can reduce the expenses for the medications.
– Providing a high throughput analytical methodology that can facilitate the processing of large number of samples in a relatively short time. This property was attributed to the use of multi-channel pipettes for efficient dispensing of the solutions, carrying out the analytical reaction in 96-well plates (as reaction vessels), and measuring the color signals in the 96 wells at ~ 30 seconds by the plate reader.
– Although the proposed assay was developed and validated for OLM, however, it is also anticipated that the same methodology could be used for essentially any analyte that can exhibit CT reaction.
limit of detection
limit of quantification
relative standard deviation
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group No. RGP-VPP-065. As well, the authors are grateful to Dr. Hamdy M. Abdel-Rahman (Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Egypt) for performing the molecular modeling.
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